JP2003525880A - Aspirin-induced lipid mediator - Google Patents

Abstract

(57) Abstract Aspirin-induced lipid mediators (ATLMs) useful for treating or preventing inflammation associated with various diseases, including ischemia, are disclosed.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Numerous reports over the past 25 years show that supplementing dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) with flaxseed oil, canola oil or fish oil has beneficial effects on human disease and experimental animals. (1. De Caterina, R., S. Endres, SD
Kristensen, and EB Schmidt, 1993. n-3 Fatty Acids and Vascular Disea
se. Spriger-Verlag, London and 2. Lands, WEM, eds., 1987. Proceedings o
f the AOCS Short Course on Polysaturated Fatty Acids and Eicosanoids. Am
erican Oil Chemists' Society, Champaign, IL) for their potential anti-thrombotic, immunomodulatory, and anti-inflammatory responses associated with arteriosclerosis, arthritis, and asthma as well as anti-tumor and anti-metastatic effects. Active discussions are also included (Ref. 1 and Iigo, M., T. Nakagawa, C. Ishikawa, Y. Iwahori, M. Asamoto, K.
Yazawa, E. Araki, and H. Tsuda. 1997. Inhibitory effects of docosahexaen
oic acid on colon cartinoma 26 metastasis to the lung. Br. J. Cancer 75
: 650-655). Their prophylactic potential in cardiovascular disease has recently been shown to be the major ischemic diet ω-3 PUFAs, eicosapentaenoic acid (C20: 5ω-3; EPA) and docosahexaenoic acid (C22: 6ω-3; DHA). It was supported by the finding that it had a dramatic effect on induced ventricular fibrillation and could protect against sudden cardiac death in dogs (4. Billman, GE e.
t al. 1999 Prevention of sudden cardiac death by dietary pure ω-3 polyu
nsaturated fatty acids in dogs. Circulation. 99: 2452-2547). Child nutrition,
The emergence of such potential preventive and / or therapeutic effects of omega-3 PUFA supplementation in cardiovascular disease and mental health has been the basis for food intake recommended by the International Workshop (5. Simopoulous, AP et al., 1999. Workshop on the
essentiality of and Recommended Dietary Intakes for Omega-6 and Omega-3
Fatty Acid. J. Am. Coll. Nutr. 18: 487-489). Also, Gruppo Italiano per
lo Studio della Sopravvivense nell 'Infarto Miocardio (GISSI) Prevenzio
ne trial includes 1 g of omega-3 PUFA daily in addition to recommended preventive measures including aspirin
We evaluated the effect of ω-3 PUFA supplementation in ingested survivors of myocardial infarction> 11,300 (n
= 2,836), and reported an important benefit with reduced cardiovascular death (6. Marchiol).
oi, R. 1999. Dietary supplementation with n-3 polyunsaturated fatty acid
s and vitamin E after myocardial infarction: results of the GISSI-Preven
zione trial. Gruppo Italiano per lo Studio della Sopravvivense nell 'Inf
arto miocardioco. Lancet, 354: 447-455). However, research on neural tissue (
In all studies (including Parkinson's disease and Alzheimer's disease and others known to be involved in brain inflammation), the cellular and molecular mechanisms of the protective effect of food ω-3 have largely been unexplained to date.

The action of C20: 5, the main lipid of fish oil, is as follows: (a) Arachidonic acid ((C20: 4ω-6; AA)
The inhibition of the conversion of erythrocytes to proinflammatory eicosanoids (ie prostaglandins [PGs] and leukotrienes [LTS]); (b) as an alternative substrate to produce weak 5-series LTS; and / or (c) antithrombotic effect Based on the conversion by cyclooxygenase (COX) to prostanoids (ie PGI ₃ ) with potency comparable to their 4-series PG counterparts (refs. 1, 3 and 4). These and other proposed explanations are generally accepted due to the lack of molecular evidence in vivo and the high concentration of ω-3 PUFA required to obtain the putative “beneficial effect” in vitro. It did not come (references 1-5).

Although a pro-inflammatory role for LT and PG is recognized, other eicosanoids from arachidonate, lipoxins (LXs) and their endogenous analogs, aspirin-induced 15-epimer LXs (ATLs), are PMN-mediated. There is new evidence that it is a potent inverse regulator of injury and acute inflammation (7. Weissmann, G. 1991. Aspirin. Sci. A
m. 264: 84-90, 8. Marcus, AJ 1999. Platelets: their role in hemostasi
s, thrombosis, and inflammation. In Inflammation: Basic Principals and C
linical Correlates. JL Gallin and R. Snyderman, Ed., Lippincott William
s & Wilkins, Philadelphia, 77-9; 9. Claria, J. and CN Serhan. 1995. As
pirin triggers previously undescribed bioactive eicosanoids by human end
otherial cell-leukocyte interactions. Proc. Natl. Acad. Sci. USA 92: 947
5-9479: 10. Serhan, CN, JF Maddox, NA Petasis, I. Akritopoulou-Zan
ze, A. Papayianni, HR Brady, SP Colgan, and JL Madara. 1995. Desig
n of lipoxinA ₄ stable analogs that block transmigration and adhesion of
human neutrophils Biochemistry 34: 14609-14615; and 11. Chiang, NK Gro
nert, CB Clish, JA O'Brian, MW Freeman, and CN Serhan. 1999, Leu
kotrieneB ₄ receptor transgenic mice reveal novel protective roles for li
poxins and aspirin-triggered lipoxins in reperfusion. J. Clin. Invest. 1
04: 309-316). C, the classical site of action for nonsteroidal anti-inflammatory drugs (NSAIDs)
It has been demonstrated that at least two isoforms of OX (COX-1 and 2) appear to play distinct physiological and pathophysiological roles in humans (12. Herschman, HR 1998, Recent progress in the cellular and mo
lecular biology of prostaglandin systhesis. Trends Cardiovasc. Med. 8: 1
45-150) Each COX isoform has dual enzymatic activities, bisoxygenase and peroxidase. Selective inhibition of COX-2 without inhibiting COX-1 results in conventional NS
Inhibition of COX-2 is the current focus of several pharmaceutical companies because it can reduce the unwanted side effects associated with AIDs (13. Needleman, P. and PC Isacson.
1997. The discovery and function of COX-2. J. Rheumatol. 24 (Suppl. 49
): 6-8). In this regard, COX-2 acetylation by the classical NSAID aspirin (ASA) inhibits prostanoid formation, whereas the acetylated enzyme is in sit
Still active in us, C20: 4-15R-hydroxyeicosatetraenoic acid (
15R-HETE), which is released and activated by activated inflammatory cells to induce 15-epimer LXs.
(14. Ching, NT Takano, CB Clish, NA Petasis, H.-H. Ta
i, and CN Serhan. 1998. Aspirin-triggered15-epi-lipoxinA ₄ (ATL) genera
tion by human leukocytes and murine peritonitis exudates: Development of
a specific 15-epi-LXA ₄ ELISA, J. Pharmacol. Exp. Ther. 287: 779-790 and
15. Xiao G., A.-L. Tsai, G. Palmer, WC Boyar, PJ Marshall, and RJ
Kulmacz, 1997. Analysis of hydroperoxide-induced tyrosyl radicals and l
ipoxigenase activity in aspirin-treated human prostaglandinH syntase-2.
Biochemistry 36: 1836-1845). Synthetic analogues of these natural topical mediators with a long biological half-life show potent anti-inflammatory effects, and AA (and / or A) to mediators with anti-inflammatory effects through modulation of signaling important in host defense. Provide evidence that cell-cell interactions can be involved in the conversion of other lipids and PUFAs (see Figure 1) (refs. 11 and 16. Clish, CB, JA O'Brien, K.).
Gronert, GL Stahl, NA Petasis, and C, N. Serhan. 1999. Local and sys
temic delivery of a stable aspirin-triggered lipoxin prevents neutrophil
recruitmemt in vivo. Proc. Nad. Acad. Sci. USA 96: 8247-8252).

SUMMARY OF THE INVENTION Although aspirin therapy inhibits prostaglandin biosynthesis without acting directly on lipoxygenase, it acetylates cyclooxygenase 2 (COX-2) to form a carbon-15 epimeric bioactive lipoxin (15- epi-LX, also called aspirin-induced LX [ATL]). The present invention provides that inflammatory exudates of mice treated with omega-3 polyunsaturated fatty acids and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells treated with ASA and having upregulated COX-2 show C20: 5ω-3 with 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-
Convert to HEPE. Each is used by polymorphonuclear leukocytes to generate distinct classes of novel trihydroxy-containing mediators, including the 5-series 15R-LX and 5,12,18R-triHEPE. These novel compounds have been shown to be potent inhibitors of transendothelial cell migration and invasion of human polymorphonuclear leukocytes in vivo (ATL analogs> 5,12,
18R-Tri HEPE> 18R-HEPE). Acetaminophen and indomethacin also show ω-3
Production of 18R-HEPE and 15R-HEPE by recombinant COX-2 as well as ω-9 and ω-9, and polyunsaturated fatty acids that act on blood vessels, brain, inflammation and blood cells (eg C18
: 3, C22: 6) and other new oxygenations are possible. These findings demonstrate a novel transcellular pathway that results in an array of bioactive lipid mediators with cell-cell interactions that affect COX-2-nonsteroidal anti-inflammatory drug-dependent oxygenation and microinflammation.
The production of these and related compounds provides the therapeutic benefits of omega-3 food supplementation with significant impact on inflammation, tissue overgrowth, and vascular disease.

Oxidation of C20: 4 by P450s in endothelial cells (ECs) also leads to 11,12-epoxyeicosatetraenoic acid, which appears to inhibit EC activation, whereas non-enzymatic oxidation of EPA is an EC adhesion molecule Can be down-regulated (17. Node, KY Huo, X,
Ruan, B. Yang, M. Spiecker, K. Ley, DC Zeldin, and JK Liao. 1999. A
nti-inflammatory properties of cytochrome P450 epoxygenase-derived eicos
anoids, Science 285: 1276-1279 and 18. Sethi, S., AY Eastman, and J.W.
Eaton. 1996. Inhibition of phagocyte-endothelium interactions by oxidize
d fatty acids: A natural anti-inflammatory mechanism? J. Lab. Clin. Med.
128: 27-38). Since PMN-vascular interactions are central to recruitment and PMN-dependent tissue injury, the local signals involved in their “cross talk dialogue” are of interest. The present invention shows that aspirin-acetylated COX-2 is in vivo
Of ASA's beneficial effects that should remain active in, but not be solely for, prostanoid inhibition, providing for the generation of specific ATLs that may be well-effected anti-inflammatory response effectors. Provide a mechanism (references 8, 12, 14). New therapeutic applications of ASA and related NSAIDs continue to emerge. However, they usually require molecular definitions to provide rationale. This includes the reported prophylactic benefit of ASA in colorectal cancer and a lower risk of a second myocardial infarction (Review 19. Levy, GN 1997. Prostaglandin H Synthases, nons.
teroidal anti-inflammatory drugs, and colon cancer.FASEB J. 11: 234-247
). In view of the qualitatively overlapping beneficial aspects attributed to food (ω) -3 PUFAs in human disease (references 1-6), the present invention provides a molecular basis for these beneficial aspects. It is directed to a novel pathway for lipid-derived signals that also serves as a marker of action.

The present invention relates to polyunsaturated fatty acids (PUFA (s)), namely C18: 3, C20: 4 and C
Methods for treating or preventing inflammation in a mammal by administration of a combination of polyunsaturated fatty acids, including 22: 6, and aspirin are described. In one aspect,
Omega fatty acids such as C18: 3 or C22: 6 and aspirin are administered at two different times. The present invention provides for the administration of a combination of omega fatty acids and aspirin to a mammal such that the mammal has arterial inflammation, arthritis, psoriasis, urticaria, vasculitis, asthma, ocular inflammation, pneumonia, pulmonary fibrosis, seborrhea. Methods for treating atopic dermatitis, purulent dermatitis, or cardiovascular disease are also described.

[0007]   In another aspect, the invention provides the following formula:

[0008]

[Chemical 25]

[0009] Or

[0010]

[Chemical formula 26]

And their stereoisomers, such as enantiomers, diastereomers, racemates, wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, such as those pharmaceutically acceptable By the administration of an anti-inflammatory drug of the natural class ASA-induced lipid (ω-3) mediator
Methods for treating or preventing inflammation in a mammal are also described. Preferred analogs include methyl, ethyl and glycerol esters. P is H (hydroxy)
Or groups with a wide variety of hydroxyl groups, such as suitable protecting groups or groups known in the art. These hydroxyl protecting groups include esters (acetate, ethyl acetate), ethers (methyl, ethyl), ethoxylated derivatives (ethylene glycol, propylene glycol) and silylated groups (TMS or TIPPS).

In another aspect, the present invention is directed to treating or preventing inflammation in a mammal by administration of an anti-inflammatory drug of the natural class of ASA-induced lipid (ω-2, ω-3 or ω-4) mediators. Compositions and methods are described in which the mediators are monohydroxylated docosahexaenoic acid (DHA) (C22: 6), ie 13-hydroxy-DHA, 14-hydroxy-DHA, 16-hydroxy-DHA, 17- Hydroxy-DHA, 19-hydroxy-DHA, or 20-hydroxy-DHA, wherein the carboxylic acid is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, such as a pharmaceutically acceptable analog thereof. It can be a functional group, like the body. Preferred analogs include methyl, ethyl, and glycerol esters. The hydroxyl groups of the monohydroxylated DHA compound can also be protected as described herein. Suitable protecting groups or groups are those known in the art. These include esters (acetate, ethyl acetate), ethers (methyl, ethyl), ethoxylated derivatives (ethylene glycol, propylene glycol) and silyl ether groups (TMS or TIPPS).

In one aspect of the invention, the compounds of the invention are substantially purified and isolated by techniques known in the art. Purified compounds generally have a purity by weight of at least about 90%, preferably at least about 95%, and most preferably at least about 99%.
%.

In yet another aspect, the invention comprises a method for treating arterial inflammation, arthritis, or cardiovascular disease in a mammal, comprising administering to the mammal one or more compounds as described above. Will be explained.

Surprisingly, it was unexpectedly found that the interaction between aspirin, COX-II and ω-3 and ω-6 fatty acids has an anti-inflammatory effect on tissues. Furthermore, this combination produces a unique compound having the above-identified formula. These compounds can be used as anti-inflammatory agents for the treatment of disease states or disease states with inflammation associated with these diseases or conditions.

Advantageously, the compounds and methods of this invention have minimal side effects. Targeting neutrophils by the compounds of the invention prevents the unique side effects associated with NSAIDs. NSAIDs
Has a wide range of biological / physiological effects, but the compounds of the invention are selective for neutrophils. Because these compounds are neutrophil-directed therapeutics to reduce inflammation, they have dramatically fewer side effects than typical NSAIDs. The compounds of the present invention are
Constipation, nephrotoxicity and gastrointestinal ulceration or bleeding have minimal, if any, undesired side effects. These benefits provide a useful alternative for patients seeking to alleviate the inflammatory condition, otherwise they may have NSAI.
You will in vain suffer one or more typical side effects associated with Ds.

DETAILED DESCRIPTION OF THE INVENTION Features and other details of the invention will be more particularly pointed out and pointed out herein in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not limitation of the invention. The main features of this invention can be used in various aspects without departing from the scope of the invention.

Abbreviations used throughout this patent application include the following and are included here for convenience. NSAID, non-steroidal anti-inflammatory drug; PUFA, polyunsaturated fatty acid; ALXR, LXA ₄
Receptor; ASA, aspirin; ATL, aspirin-induced 15-epi-LX, 15R-LX; ATLM, aspirin-induced lipid mediator; COX, cyclooxygenase I, II (isoform); EC, endothelial cells; HUVEC, human umbilical cord endothelial Cell; LC / MS / MS, liquid chromatography tandem mass spectrometry; LM, lipid-derived mediator; LO, lipoxygenase; LT, leukotriene; LX, lipoxin; PG, prostaglandin; PMN
, Polymorphonuclear leukocytes; EPA, eicosapentaenoic acid; HEPE, hydroxyeicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid; LXA ₄ , 5S, 6R, 15S-trihydroxy-7,9,13-trans-11- Cis-eicosatetraenoic acid; 15-epi-LXA ₄ , 5S, 6R
, 15R-Trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; C20: 5
(Eicosapentaenoic acid, EPA, ω-3 fatty acid); C20: 4 (Arachidonic acid, AA, ω-6
Fatty acids); and C22: 6 (docosahexaenoic acid, DHA, omega-3 fatty acids).

The present invention describes a method for treating or preventing inflammation in a mammal by administration of a combination of omega-3 (ω-3) fatty acids and aspirin. In one embodiment, the omega fatty acid, eg EPA or DHA, and aspirin are administered at two different times. The present invention also describes methods for treating arterial inflammation, arthritis, or cardiovascular disease in a mammal by administering to the mammal a combination of an omega fatty acid such as EPA or DHA and aspirin.

In another aspect, compositions and methods for treating or preventing inflammation in a mammal by administration of an anti-inflammatory drug having one of the following formulas are described. Compound 1
Is the following formula:

[0021]

[Chemical 27]

With In a preferred embodiment, the hydroxyl at carbon 15 has the R configuration. In another embodiment, the hydroxyl at carbon 15 has the S configuration. Alternatively, the hydroxyl at carbon 15 is an R / S racemic mixture.

[0023]   Compound 2 has the following formula:

[0024]

[Chemical 28]

With In a preferred embodiment, the hydroxyl at carbon 18 has the R configuration. In another embodiment, the hydroxyl at carbon 18 has the S configuration. Alternatively, the hydroxyl at carbon 18 is an R / S racemic mixture.

[0026]   Compound 3 has the following formula:

[0027]

[Chemical 29]

[0028] In one embodiment, the hydroxyl at the carbon 5 position has the S configuration, the hydroxyl at the carbon 6 position has the R configuration, and the hydroxyl at the carbon 15 position has the R configuration. In another embodiment, the hydroxyl at the carbon 5 position is
It has the R / S configuration, the hydroxyl at carbon 6 has the R / S configuration, and the hydroxyl at carbon 15 has the R / S configuration.

[0029]   Compound 4 has the following formula:

[0030]

[Chemical 30]

With In one embodiment, 5-hydroxyl has the S configuration, 12-hydroxyl has the R configuration, and 18-hydroxyl has the R configuration. In another embodiment, 5-hydroxyl has the R / S configuration, 12-hydroxyl has the R / S configuration, and 18-hydroxyl has the R / S configuration.

[0032]   Compound 5 has the following formula called 13-hydroxy-DHA:

[0033]

[Chemical 31]

With P = H (hydroxyl). In one aspect, the 13-hydroxyl has the S configuration. In another embodiment, the 13-hydroxyl has the R configuration. In yet another embodiment, the 13-hydroxyl is a racemic mixture, such as R / S
It is a three-dimensional arrangement.

[0035]   Compound 6 has the following formula called 14-hydroxy-DHA:

[0036]

[Chemical 32]

With P = H (hydroxyl). In one aspect, the 14-hydroxyl has the S configuration. In another embodiment, the 14-hydroxyl has the R configuration. In yet another embodiment, the 14-hydroxyl is a racemic mixture such as R / S
It is a three-dimensional arrangement.

[0038]   Compound 7 has the following formula called 16-hydroxy-DHA:

[0039]

[Chemical 33]

With P = H. In one aspect, the 16-hydroxyl has the S configuration. In another embodiment, the 16-hydroxyl has the R configuration. In yet another embodiment, the 16-hydroxyl is in a racemic mixture, eg in the R / S configuration.

[0041]   Compound 8 has the following formula called 17-hydroxy-DHA:

[0042]

[Chemical 34]

And where P = H. In one aspect, the 17-hydroxyl has the S configuration. In another embodiment, the 17-hydroxyl has the R configuration. In yet another embodiment, the 17-hydroxyl is in a racemic mixture, eg in the R / S configuration.

[0044]   Compound 9 has the following formula called 19-hydroxy-DHA:

[0045]

[Chemical 35]

With P = H. In one aspect, the 19-hydroxyl has the S configuration. In another embodiment, the 19-hydroxyl has the R configuration. In yet another embodiment, the 19-hydroxyl is in a racemic mixture, eg in the R / S configuration.

[0047]   Compound 10 has the following formula called 20-hydroxy-DHA:

[0048]

[Chemical 36]

With P = H. In one aspect, the 20-hydroxyl has the S configuration. In another embodiment, the 20-hydroxyl has the R configuration. In yet another embodiment, the 20-hydroxyl is in a racemic mixture, eg in the R / S configuration.

In compounds 1 to 10, R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug. Preferred analogs include methyl, ethyl and glycerol esters.

It should be understood that in compounds 1 to 10, when referring to the “hydroxyl” stereochemistry, it is typical and such terms are meant to also include protected and free hydroxyl groups. is there.

The hydroxyls in compounds 1-10 can be protected by various protecting groups known in the art. One of ordinary skill in the art can readily determine which protecting groups are useful for protecting the hydroxyl groups. Standard methods are known in the art and are more fully explained in the literature. For example, a suitable protecting group can be selected by one of ordinary skill in the
een and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and
Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups include methyl and ethyl ethers, TMS or TIPPS groups, acetate or propionate groups and glycol ethers such as ethylene glycol ethers and propylene glycol derivatives.

For example, one or more hydroxyl groups can be treated with a mild base such as triethylamine in the presence of acid chloride or silyl chloride to promote the reaction between the hydroxyl ion and the halide. Alternatively, the alkyl halide can be reacted with hydroxyl ions (generated by a base such as lithium diisopropylamide) to promote ether formation.

It should also be understood that in compounds 3 and 4 not all hydroxyl groups need be protected. One, two or all three hydroxyl groups may be protected. This can be accomplished by stoichiometric selection of the reagents used to protect the hydroxyl groups. Methods known in the art such as HPLC
, LC, flash chromatography, gel permeation chromatography, crystallization,
Distillation and the like may be used to separate mono-, di-, or tri-protected hydroxy compounds.

It should be understood that each of the above-identified compounds has one or more chiral centers. It is to be understood that this invention includes all stereochemical forms of each compound, eg enantiomers, diastereomers and racemates.

The term “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refers to carboxylic acid salts, amino acid-loaded salts, esters, amides, and prodrugs of compounds of the present invention. Represents, where possible, like the zwitterionic forms of the compounds according to the invention, they have no undue toxicity, irritation, allergic reactions, etc., and come into contact with the tissue of the patient, within sound medical judgment. It is suitable for the intended use, has a reasonable benefit / risk ratio, and is effective for its intended use. The term "salt" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts are prepared either in situ during the final isolation and purification of the compound or by reacting the appropriate organic or inorganic acid with the purified compound in the free base form separately and isolating the salt formed. You may. These include cations based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, etc., as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, triethylamine, ethylamine, etc. It may include, but is not limited to, non-toxic ammonium, quaternary ammonium, and aminating cations. (For example, Berge SM et al., “Pharmaceutical Salts,” J. Pharm. Sci., 19 which is incorporated herein by reference.
77; 66: 1-19).

The term “prodrug” refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. T. for a full explanation
Higuchi and V. Stella, “Pro-drugs as Novel Deliverly Systems,” Vol. 1
4 of the ACS Symposium Series and Bioreversible Carriers in Drug Des
ign, Edward B. Roche, American Pharmaceutical Assoiciation and Pergamo
n Press, 1987, which are incorporated herein by reference. As used herein, a prodrug is metabolized when administered in vivo,
A compound that would otherwise be transformed into the form of a biologically, pharmaceutically or therapeutically active compound. To produce a prodrug, the pharmaceutically active compound will be modified and metabolic processes will regenerate the active compound. Prodrugs may be designed to alter the metabolic stability or transport characteristics of the drug, to protect against side effects or toxicity, to improve the taste of the drug, or to alter other characteristics or properties of the drug. Good. Once a pharmaceutically active compound is identified, knowledge of in vivo pharmacokinetic processes and drug metabolism is used to
Those skilled in the pharmaceutical arts can design prodrugs of compounds [eg, Nogrady (1985) Medical Chemistry A Biochemical Approach, Oxford Univer
sity Press, New York, pp. 388-392]. Conventional methods for the selection and manufacture of suitable prodrug derivatives are described, for example, in “Design of Prodrugs,
H. Bundgaard, Ed., Elsevier, 1985. Suitable examples of prodrugs include the methyl, ethyl and glycerol esters of the corresponding acids. It would be desirable to be able to become the parent compound, the natural hydroxyl functionality.

The compounds of the present invention may be formulated into pharmaceutical compositions as described below. In a preferred embodiment, the compounds may be administered in a sustained release composition over an extended period of time. Sustained release compositions are known in the art and one of ordinary skill in the art can formulate acceptable compositions based on the parameters generally accepted in the art. In the most preferred embodiment, the glycerol ester may be used in the treatment of inflammatory conditions described herein as a sustained release composition known in the art, ie a transdermal patch. A suitable method for making a transdermal patch is described in US Pat. No. 5,814,59.
9,5,846,974 or 4,201,211 which are incorporated herein by reference. More specifically, the compounds are Ciba-Geigy and Alza Corporati
It can be delivered transdermally using the type of patch technology available from on.
The pharmaceutical composition of the invention may be administered to warm-blooded animals such as humans intermittently or at a stepwise, continuous, continuous or controlled rate. Moreover, the suitable time of day for which the pharmaceutical composition is administered and the number of administrations per day may vary. Administration is preferably such that the active ingredients of the pharmaceutical composition can interact with the inflammatory condition.

In yet another aspect, the invention comprises a method for treating arterial inflammation, arthritis, or cardiovascular disease in a mammal, comprising administering to the mammal one or more of the compounds described above. Will be explained.

The term “subject” as used herein refers to any living organism in which an immune response, eg an anti-inflammatory response, is elicited. The term subject refers to non-human primates such as humans, chimpanzees and other apes and monkey species; livestock such as cattle, sheep, pigs, goats and horses; pets such as dogs and cats; mice, rats and guinea pigs. Including but not limited to laboratory animals such as. Such terms do not mean a particular age or sex. Thus, adult and newborn infants as well as fetuses, whether male or female, are intended to be covered.

The term “mammal” as used herein refers to a living organism capable of eliciting an immune response against an antigen. The term subject refers to non-human primates such as chimpanzees and other apes and monkey species, sheep, pigs,
Including but not limited to goats, horses, dogs, cats, mice, rats and guinea pigs and the like.

The pharmaceutical composition of the present invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of the anti-inflammatory drug of the present invention. A "therapeutically effective amount" refers to an amount that is effective over the dosage and duration required to reduce or prevent the desired therapeutic result, eg, inflammation associated with various disease states. The therapeutically effective amount of an anti-inflammatory drug may vary depending on such factors as the disease state, age, sex and weight of the individual, and the ability of the anti-inflammatory drug to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. "
A "prophylactically effective amount" is an amount that is effective over the dose and duration of administration necessary to achieve the desired prophylactic result. Generally, a prophylactic dose is used in a subject prior to or at an early stage of the disease. , A prophylactically effective amount will be less than a therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (eg,
Therapeutic or prophylactic response). For example, a single bolus dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Good. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit doses for the mammalian subject to be treated; each unit being a pharmaceutical carrier required With a predetermined amount of active compound calculated to produce the desired therapeutic effect. Details regarding the dosage unit form of the present invention can be found in (a) the specific properties of the active compound, and the particular therapeutic or prophylactic effect sought to be obtained, and (b) for the treatment of individual susceptibility. Subject to and directly dependent on the limitations inherent in the art of formulating such active compounds.

A typical, non-limiting range for a therapeutically or prophylactically effective amount of an anti-inflammatory agent of the present invention is 0.
It is 1-20 mg / kg, more preferably 1-10 mg / kg. It should be noted that dose values may vary depending on the type and severity of the condition to be alleviated.
For any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or administering the composition. It should be further understood that, and the dosage ranges disclosed herein, are merely exemplary and are not intended to limit the scope and practice of the claimed compositions. The anti-inflammatory compounds of the invention, eg compounds 1 to 10, can be incorporated into pharmaceutical compositions suitable for administration to a subject. Generally, the pharmaceutical composition comprises an anti-inflammatory drug of the invention and a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any and all physiologically suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Including. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Pharmaceutically acceptable carriers may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the preservative or effectiveness of the anti-inflammatory agent.

The anti-inflammatory agents of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration. Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate and the like. Sodium chloride can be used to modify the toxicity of the solution at concentrations of 0-300 mM (optimally 150 mM for liquid dosage forms). Cryoprotectants, mainly 0-10
% Sucrose (optimally 0.5-1.0%) may be included in the lyophilized dosage form. Other suitable cryoprotectants include trehalose and lactose. Bulking agents, primarily 1-10% mannitol (optimally 2-4%), may be included in the lyophilized dosage form. Stabilizers, primarily 1-50 mM L-methionine (optimally 5-10 mM) may be used both in liquid and lyophilized dosage forms. Other suitable bulking agents include glycine, arginine and may be included such as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Polysorbate 20 and BR for additional surfactants
Examples include, but are not limited to, IJ surfactants.

The compositions of the present invention may be in a variety of shapes. These include liquid, semi-solid and solid dosage forms such as, for example, liquid solutions (eg injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic effect. Generally preferred compositions are in the form of injection or infusion solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the anti-inflammatory drug is administered by intravenous infusion or injection. In another preferred embodiment, the anti-inflammatory drug is administered by intramuscular or subcutaneous injection. In the most preferred embodiment, the anti-inflammatory drug is administered orally.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injection
If desired, by incorporating one or a combination of the components described above in a suitable solvent and the required amount of the active compound (eg, antigen, antibody or antibody portion), followed by filtration and sterilization. It may be manufactured.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients as noted above. In the case of sterile, lyophilized powders for the production of sterile injectable solutions, the preferred method of preparation is to obtain a powder of the active ingredient and a sterile powder of any of the additional desired ingredients derived from their sterile-filtered solution above. Vacuum drying and spray drying. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, for example, monostearic acid and gelatin.

The anti-inflammatory drug of the present invention may be administered by various methods known in the art. As will be appreciated by those in the art, the route and / or mode of administration will vary depending on the desired result. In certain embodiments, the active compounds may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems. Ethylene vinyl acetate,
Biodegradable, biocompatible polymers may be used, such as polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the manufacture of such formulations are either patented or known to those skilled in the art. For example, Sustained and Controlled Release Drug Delivery Systems, JR R
In certain embodiments, see obinnson, Ed., Marcel Dekker, Inc., New York, 1978, the anti-inflammatory agents of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients if desired) may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the subject. For oral therapeutic administration, the compound is incorporated with excipients and ingestible tablets,
It may be used in the form of buccal agents, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Administration of the compounds of the present invention other than parenterally requires that the compound be coated with or co-administered with a material that inhibits inactivation.

As mentioned above, compounds 1 to 10 according to the invention and their pharmaceutically acceptable analogues have use in the prevention and treatment of clinical conditions involving the COX enzyme which causes inflammatory conditions. For example, the ability of the compounds of the invention to interact with COX enzymes helps them to prevent and treat convulsive conditions, allergic conditions, inflammatory conditions associated with conditions involving platelet aggregation, and more commonly recognized inflammatory conditions. .

Examples of convulsive states include those containing smooth muscle tissue, particularly asthma (including idiopathic bronchial asthma), airway smooth muscle contraction such as bronchitis, and coronary spasm (those associated with myocardial infarction). Includes, with or without left-sided heart failure leading to cardiac asthma), ischemia-induced myocardial damage, and arterial smooth muscle contractions such as convulsions or seizures (which may cause central nervous system abnormalities) There are things. Other examples include inflammatory bowel disease, bowel disease caused by abnormal rectal muscle contractions such as the condition known as rectal spasm and mucocolitis.

Examples of allergic conditions include exogenous asthma, allergic skin diseases with wholly or partially allergic origin such as eczema, allergic enteropathy (including pediatric steatorrhea), hay fever (which is , Or instead may affect the upper respiratory tract), allergic rhinitis, and allergic conjunctivitis.

Examples involving platelet aggregation include strokes with a wholly or partially thrombotic origin,
Those due to thrombosis are included, including coronary thrombosis, phlebitis and venous thrombosis (the latter two conditions are probably also associated with inflammation).

Examples of inflammatory conditions include lung, joint, eye, intestine, skin, and heart conditions; particularly those involving infiltration of leukocytes into inflamed tissue. As an inflammatory lung condition, asthma,
Adult respiratory distress syndrome, bronchitis and cystic fibrosis (in addition or in the alternative, may include intestines or other tissues). Inflammatory joint conditions include rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gout arthritis, and other arthritic conditions. Inflammatory eye conditions include uveitis (including iritis) and conjunctivitis. Inflammatory bowel conditions include Crohn's disease, ulcerative colitis and distal proctitis.

Inflammatory skin diseases include dermatitis, allergic or irritant contact skin, including cell proliferation related diseases such as psoriasis, eczema, eczema-like dermatitis such as atopic and seborrheic dermatitis. Inflammation, eczema craquelee, photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, and congestive dermatitis. Inflammatory skin diseases include ulcers and erosions due to trauma, burns, bullous diseases, or ischemia of the skin or mucous membranes; some forms of ichthyosis; epidermolysis bullosa; hypertrophic scars and keloids; essential aging and Examples include, but are not limited to, photoaging; frictional blistering caused by mechanical shearing of the skin; and skin atrophy resulting from topical use of corticosteroids. In addition, inflammatory skin conditions include cheilitis, cracked lips, nasal irritation and inflammation of the mucous membranes such as vulvovaginitis.

Inflammatory conditions of the heart include myocardial infarction injury. Other inflammatory conditions include skin necrosis in chronic inflammation, endotoxin shock, smooth muscle proliferative disorders (eg restenosis after angioplasty), and tissue rejection after transplant surgery.

Other examples of disease states associated with inflammation include those described below. All examples of disease states presenting inflammatory conditions described throughout this specification are encompassed by the inventive concept of treating inflammation. Moreover, these lists of inflammatory conditions are typical and not exhaustive. One of ordinary skill in the art will appreciate that PMNs and / or leukocytes may be included in the concept for alleviating an inflammatory condition that is locally increased relative to baseline due to injury, injury, or an irritant to the subject's physiology. You will recognize the inflammatory condition.

Accordingly, the present invention provides a method for the prevention or treatment of a clinical condition in a subject such as a convulsive condition, an allergic condition, a condition involving platelet aggregation, or an inflammatory condition. Treatment includes the administration of a therapeutically effective amount of one or more compounds 1 to 10 of the invention, or pharmaceutically acceptable analogs described herein. The invention further comprises
Prevention or treatment of neurodegeneration or dementia associated with HIV infection in a subject comprising administration of a therapeutically effective amount of one or more Compounds 1-10 of the invention, or a pharmaceutically acceptable analog described herein. To provide a method.

In one aspect, the anti-inflammatory agents of the present invention may be incorporated into shampoos or body cleansing products for cleansing the scalp and / or body, such as soaps. The use of these compounds in shampoo or soap products may be used to treat psoriasis, seborrheic dermatitis, purulent dermatitis and dandruff. In addition, the anti-inflammatory agents of the invention, such as compounds 1-10, and combinations thereof, are for treating the diseases and sunburns described above, venom ivy, dermatitis, and for slowing the growth of metastatic cancer. May be used for topical lotion. Alternatively, the compounds of the present invention may be used to treat Alzheimer's disease, where the action of anti-inflammatory drugs is known to help reduce the long-term effects of plaque formation. In an alternative embodiment, the compounds of the invention may generally be used as an aerosol or spray to treat airway inflammation such as, for example, bronchitis, asthma, pneumonia, emphysema, and upper respiratory tract disease. .

Supplementary active compounds may be incorporated into the compositions. In certain embodiments, the anti-inflammatory agents of the invention are formulated and / or administered with one or more additional therapeutic agents useful in the treatment of diseases in which inflammation is detrimental. For example, the anti-inflammatory agents of the invention may be formulated and / or administered with other targets, such as one or more additional anti-inflammatory agents that bind to the receptor. Furthermore, one or more anti-inflammatory agents of the present invention may be used in combination with two or more of the above therapeutic agents. Such combination therapies may conveniently use lower dosages of the therapeutic agents administered, thus avoiding the toxicity or complications that may be associated with various monotherapies.

Surprisingly, the interaction between aspirin, COX-II and omega-3 fatty acids has been unexpectedly found to have an anti-inflammatory effect on tissues. further,
This combination yields a unique compound having the formula identified above. These compounds have anti-inflammatory effects and can be used as anti-inflammatory agents in the treatment of disease states with inflammation associated with these diseases or conditions.

Two classes of eicosanoids, LT and certain PGs, mediate important effects associated with human disease. Proinflammatory effects of LT and PG have been demonstrated (Fig. 1
Reference), the present invention provides novel evidence that previously unknown LMs and their stable analogs have potent counterregulatory effects in PMN-mediated tissue damage and acute inflammation. In line with these principles, p450 oxidation of arachidonic acid in endothelial cells (EC) leads to 11,12-EET displaying an anti-inflammatory effect that inhibits leukocyte adhesion molecules, and also non-enzymatic oxidation of EPA. It is known to produce an as yet unidentified product that down-regulates EC.

The present finding is that arterial inflammation, arthritis, cardiovascular disease, senile inflammatory disease, irritable intestinal (colon), erysipelas, eczema, psoriasis, urticaria, vasculitis, AIDS with inflammation, ocular disease inflammation,
It is provided that the integrated host response, recognized as a human disease such as asthma, pneumonia, pulmonary fibrosis, reflects to some extent the overall balance between "pre" and "anti" inflammatory signals. Between such signals, the role of LM remains unproven. This is probably because these products are often rapidly produced by transcellular biosynthesis and act short-lived locally. In this regard, arachidonic acid-derived lipoxin (LX) and recently demonstrated aspirin-induced LX (ATL) and metabolically stable analogs, whose long in vivo half-life promotes beneficial effects in vivo. Because of this, I have been interested.

ASA induces the formation of epimeric forms of naturally occurring bioactive eicosanoids (
Therefore, we tested the concept that NSAIDs promote the formation of new mediators from ω-3 PUFAs. The inflammatory exudate formed in the pouch by injecting TNF-α and ω-3 and ASA into the mouse air pouch on the plate (2 h) produced several novel compounds (Fig. 4). These mice were fed a standard rodent diet containing 0.26% ω-3 PUFA. LC / MS / MS analysis of leachate-derived material showed monohydroxy acids, ie 18-hydroxy-EPA (18-HEPE) and 5-, as shown in the selected ion chromatogram from the results obtained at m / z 317 (Figure 4). Shows HEPE, which are synthetic 5S-HEP
Eluted with new trihydroxy-containing compounds from E and C 20: 5. LC retention times and MS / MS spectra (Figures 4B and 4C) gave product ions consistent with the structure shown in the respective inset, m / z 317 = (MH) -H2O-CO2. A diagnostic ion consistent with 18-HEPE identification is present at m / z 259 (Figure 4B), and 5-HE
PE was present at m / z 115 (Fig. 4C). These criteria were used throughout the identification. The stereochemistry of the 18 carbon alcohol was demonstrated for leachate-derived 18-HEPE using a chiral column, and the reference 18-HEPE was produced biosynthetically using B. megaterium (see Figure 5, Materials and Methods). Please refer). This microbial monooxygenate fatty acid converts, for example, C20: 4 into 18R-HEPE (22. Capdevila, JH, S. Wei, C. Helv
ig, JR Falck, Y. Belosludtsev, G. Truan, SE Graham-Lorence, and JA
Peterson, 1996. The highly stereoselective oxidation of polynsaturated
fatty acids by cytochrome P450 BM-3. J. Biol. Chem. 271: 22663-22671 ； an
d 23. Ruettinger, RT and AJ Fulco. 1981. Epoxidation of unsaturated
fatty acids by a soluble cytochrome P-450-dependent system from Bacillus
megaterium. J. Biol. Chem. 256: 5728-5734). The alcohol configuration at position 18 proved to be> 98% R. These findings indicate that the mouse inflammatory exudate contacts C20: 5, ω-3 and ASA in vivo, a product also identified in human PMNs, the 5-lipoxygenase pathway 5-series 5S-HEPE, and the formation pathway. Has been shown to produce a determined 18R-HEPE (see below) (24. Lee, TH, J
.M. Menica-Huerta, C. Shih, EJ Corey, RA Lewis, and KF Austen. 19
84. Characterization and biologic properties of 5,12-dihydroxy derivativ
es of eicosapentaenoic acid, including leukotriene B5 and the double lip
oxygenase product. J. Biol. Chem. 259: 2383-2389). Air pouch inflammatory exudate cells from ASA- and EPA-treated mice contained predominantly PMNs (see Figure 4) and were 25-50% less than in exudates formed with TNF-α alone (n = 3, (See Figure 6). Also, these leachates were essentially equivalent to 18R- when activated with the ionophore A ₂₃₁₈₇ (4 μM).
HEPE (10.2 ± 4.3 ng / 10 ⁴ cells) and 5S-HEPE (10.9 ± 2.9 ng / 10 ⁴ cells) were generated. FIGS. 4A-D show inflammatory exudates from mouse dorsal pouch treated with aspirin to generate novel compounds shown by LC / MS / MS; TNF-α-induced leukocyte exudates were ASA (500 μg / air pouch). At 3.5 h) and EPA (300 μg / 4 h in air sac) for 6 h, and contained 2.3 +/− 0.5 × 10 ⁶ white blood cells / sac (see methods).

Evidence for new trihydroxy-containing products was also obtained in these inflammation exudates (
(Figure 4D). The ions present in MS / MS are consistent with the 20: 5 derived trihydroxy-containing product, the parent ion at m / z 349- (M = H), and the structurally significant ions present at m / z 291 and 196. There are certain product ions, which are consistent with the fragmentation shown in the inset (Fig. 4D). Also, the 270 nm UV absorbance maximum, which is indicative of conjugated trienes, suggests that 18R-HEPE and tri-HEPE are biosynthetically related, along with m / z 291 (C17-C18 cleavage) and 20-carbon structure. did.

It was intriguing to find out whether these novel compounds were also produced by human cells. To this end, with IL-1β or hypoxia (data not available)
Human ECs known to induce COX-2 were pulsed with EPA, treated with ASA, and the extract material subjected to LC / MS / MS analysis (Figure 7A). Selected ion monitoring at m / z 259 revealed that HUVECs treated with ASA convert EPA to 18R-HEPE (Figure 7A). HMVECs processed by ASA and EPA are 18-HEPE (10.
6 ng / 10 ⁴ cells) and 15-HEPE were generated (6 ng / 10 ⁶ cells) (n = 2, 4 measurements;
No data). These observations implicate COX-2's involvement in the production of these compounds, demonstrating recombinant human COX-2 exposure to ASA and ω-3 PUFAs and revealing clinical significance (Fig. 8 and Table 1).

As shown in Table 1, linoleic acid (C18: 2) was 13-hydroxy-9Z, 11E-octadocadienoic acid (13-HODE; n-5 oxygenated) and 9-HODE (ω-9). , Which were greatly reduced by ASA but did not disappear completely. AA was converted to 15R-HETE (n-5) and 11R-HETE (n-9), consistent with earlier findings. ASA caused the appearance of lipoxygenase activity that turned to 15R-HETE production by acetylated COX-2, which did not appear to affect 11R-HETE formation (Table 1 and Figure 8). 11R-
HETE is the major product of EPA and COX-2, with small amounts of 15R-HETE (n-5) and 1
With 8R-HEPE (n-2). The ^1-14 C-labeled EPA precursor - was used to ascertain the relevant product (n = 3, FIG method 2). 18R-HEPE of ASA acetylation of COX-2 (Fig. 8, identification of each new reaction product, displayed by each mass spectrum)
About 2-fold increase of (n-2) and> 85% decrease of 11R-HETE (ratio of positional oxygenation by C20: 5 was 1: 1: 0.3, 18R-15R> 11R) . Together, they implied that the acetylated COX-2 of ECs (Fig. 7) is the major source of 18R-HEPE and 15R-HETE.

Interestingly, unlike the case of the isolated COX-2 product, 11R-HEPE (C20: 5
Origin) and 11R-HETE (from C20: 4) were not major products of vascular ECs (Table 1 and Figure 7). These oxygenations are blocked by selective COX-2 inhibitors,
And only 18R-HEPE formation from EPA seemed to escape inhibition (Table 1). These results show that ω-3, as exemplified by cytosine-driven acute inflammation (FIGS. 4 and 6).
In addition to PUFA (ie EPA; C20: 5, ω-3), ASA treatment at the local site of inflammation
It was suggested that OX-2 could convert EPA to 18R-HEPE and 15-HEPE.

Human PMNs convert ASA-induced, COX-2-derived 15R-HETE to 15-epi-LXA ₄ (reference document 9), and EPA induces human leukocytes and mass macrophages (26. Hill, DJDH, Griffi
ths, and AF Rowley. 1999. Trout thrombocytes contain 12-but not 5-lipo
xygenase activity. Biochim. Biophys. Acta 1437: 63-70) 5-series LX (2
Five. Serhan, CN, PY Wong, and B. Samuelsson. 1987. Nomenclature of lip
oxins and related compounds derived from arachidonic acid and eicosapent
aenoic acid. Prostaglandins 34: 201-204), so that activated human PMNs involved in phagocytosis are acetylated COX-2-derived C20: 5, ω-3 product, 18R-HEPE and 1
Considered handling 5R-HETE. Phagocytosis-stimulated, zymosan (STZ) treated serum showed utilization of acetylated COX-2 C20: 5 derived products and conversion of tri-hydroxy-sequential EPE into two classes, m / z 31749.5 Basal peak molecular ions of these products were again measured by selective ion monitoring at (MH)-(Figure 7B).
. One shown in FIG. 7C is essentially the same MS / MS as that observed in FIG. 4D of mouse cells.
MS, consistent with the 5,12,18R-tri-HEPE structure shown in m / z 305, 233, 195, and 291 (identifying ions in the inset as in FIG. 4D9 (FIG. 7C)).

The product is an 18R-hydroxy-containing “LTB ₅ -like” structure (see FIG. 7D, inset). Indeed, the isolated 18R-HEPE was converted to several compounds, including this product, when incubated with activated PMNs as described above. Also, synthetic LTB ₅ incubated at pH 8.0 with B. megaterium homogenate and NADPH to promote hydroxylation (ref. 23) showed 18R alcohol groups as obtained from human PMNs shown in FIG. 7C. Was converted to the trihydroxy product (n = 3) with m / z ions characteristic of the presence of (FIG. 5). These independent lines of evidence are
s uptakes 18R-HEPE, which inserts molecular oxygen and is converted in the subsequent step by their 5-lipoxygenases to 5-hydro (peroxy) -18R-DiH (p) EPE, and the precursor We showed epoxide formation to 5,12,18R-tri-HEPE (18R-containing LTB ₅ -like product), which appears to have the stereochemistry of LTB ₅ while retaining 18R chirality.

In a similar biosynthetic fashion, 15R-HEPE was converted by PMNs via 5-lipoxygenation to 5-series LTB ₅ analogs (FIG. 7D), which also retain the C15 configuration. From its MS / MS, the major ion found in the MS / MS spectrum, m /, consistent with the 15S-containing LX ₅ structure (5-series) observed in the endogenous EPA source of trout macrophages.
We obtained z305, 233, and 251, ie 15-epi-LTA ₅ . In this case, the chirality of the precursor 15R is retained by human PMN, is a 5-series omega-3 analogs of 15-epi-LXA _4, 15-epi-LTA ₅ is obtained (FIG. 7D). 5-lipoxygenation-mediated conversion of activated PMNs to both 18R- and 15R-HEPE was associated with decreased LTB ₅ formation (
No data). Taken together, these results show that isolated human ECs and PMNs (Figure 7
) Indicates that it is possible to generate the novel products observed in the inflammation exudate (Figures 1-4 and Tables 1 and 2).

Transendothelial cell migration is an important event in PMN recruitment and inflammation, and is a recognized site of action for traditional anti-inflammatory therapies (27. Cronstein, BN, SC Kimmel, RI Levin.
, F. Martiniuk, and G. Weissmann. 1992. A mechanism for the antiinflamma
tory effects of corticosteroids: The glucocorticoid receptor regulates l
eukocyte adhesion to endotherial cells and expression of endotherial-leu
kocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc.
Natl. Acad. Sci. USA 89: 9991-9995). Endogenous lipid mediators that can regulate these cell-cell interactions are of interest. Therefore, 5,1 in human PMN migration
2,18R-tri-HEPE and precursor 18R-HEPE were evaluated. Both compounds show LTB ₄ stimulated P with an apparent IC _{50 of} 5-50 nM for 5,12,18R-tri-HEPE and an IC ₅₀ > 1.0 μM for 18R-HEPE.
Inhibited MN transendothelial cell migration (Fig. 9A). Thus, the new 5-series members, 18R-carrying bird HEPE and 18R-HEPE, were tested simultaneously for direct comparison,
It inhibited PMN migration like 15-epi-LXA ₄ and omega and analogs (FIG. 10A).
, Tables 1 and 2). Their potency is 15-epi-LXA ₄ , stable analogue> 5,12,18R-
The bird HEPE was> 18R-HEPE.

A G protein-coupled receptor for LTB ₄ has been identified (28. Yokomizo, TT Izumi, K.
. Chang, T. Takuwa, and T. Shimizu. 1997. A G-protein-coupled receptor f
or leukotrieneB4 that mediates chemotaxis Nature 387: . 620-624), and to determine whether to interact with human LTB ₄ receptors to these 18R- containing product inhibits PMNs, this receptor It was cloned from the reported sequence (ref. 11) and stably expressed in HEK293 cells for competitive binding experiments. (Fig. 10B)
. The homoligand LTB ₄ competed effectively (IC ₅₀ -2.5 nM). 18R-HEPE did not compete, while LTB ₅ and 5,12,18R-tri HEPE competed together in the order LTB ₅ > 5,12,18R-tri HEPE.

Although 5,12,18R-tri-HEPE and related structures (ie LTB ₅ ) are substantially less effective than 4-series LTB _{4 in} agreement with the reduced PMN activity of LTB ₅ , [ ³ H ] The potency of replacing LTB ₄ was in the range of synthetic LTB ₄ receptor antagonists currently available (data not shown). These findings indicate that 5,12,18R-tri-HEPE, for the total synthesis of dampers of LT-mediated reactions in vivo, as well as a novel class of receptor antagonists, if produced in appropriate amounts in the microenvironment. It is suggested that it becomes a bio-template.

Low levels (100 ng) of 5,12,18R-tri-HEPE were injected intravenously in the tail, and mice were treated with equal doses for direct comparison, like the 15-epi-LX stable analog. It was a potent inhibitor of PMN infiltration into the dorsal air pouch (Fig. 10C). 18R-HEPE also has some activity in vivo (<5,12,18R-tri-HEPE), but is significantly less effective at transendothelial cell migration of isolated human PMNs, apparently recombinant at these concentrations. It did not interact with the LTB ₄ receptor.

Other widely used NSAIDs (ie, acetaminophen and indomethacin) were also recombinant COX-2 and C20 as in Table 1 to see if they alter conversion to HEPE: 5 were tested (Table 2). Each is 11-HEPE> 9
5% inhibition. Interestingly, 18R-HEPE and 15R-HETE formation persisted (~ 1: 1 ratio) in the presence of 2 mM of either acetaminophen or indomethacin, whereas levels of 15R- and 18R-HEPE were inhibitory. 1/3 of those levels in the absence of
It decreased to 1/8 (n = 3). These findings indicate that the oxygenation of omega-3 fatty acids to R-containing monohydro (peroxy) -containing products is not limited to ASA treatment and arachidonate. In fact, C18: 2, C18: 3 and C22: 6 were also converted by the NSAID-COX-2 complex into new reaction products (see Figures 11A, B and C). Thus, these commonly used NSAIDs and selective COX-2 inhibitors (Tables 1 and 2) still allow PUFA oxygenation by activated ECs exposed to NSAIDs, with drug and COX-2 at the site of inflammation.
The degree of interaction of the PUFAs allows the generation of newly oxygenated forms of PUFAs.

Despite reports of possible beneficial effects of ω-3 PUFAs (ie C20: 5) in humans, oxygen by COX-2 produces novel bioactive compounds in humans or isolated cells It has not been examined about conversion. In fish, C20: 5
And C20: 4 are both mobilized in macrophages and platelets to induce PG, LT
, And LX, producing essentially equal and abundant 5- and 4-series eicosanoids (ref. 26). The present invention provides that inflammatory exudates of mice treated with ASA and EPA produce novel compounds (Figure 4), which are also produced by human ECs, recombinant COX-2, and PMNs (Figure Five). Ω as a nutritional supplement (references 1-6)
Assuming ingestion of -3 PUFA in milligram to gram quantities, large areas of microvessels carry up-regulated COX-2, as observed in this experiment (Figure 4-8).
) Significant amounts of ECs and neighboring cells transform EPA in the local microenvironment. COX-2-NSAID-dependent conversion of these ω-3 PUFAs is likely elevated in inflammatory or diseased tissues where COX-2 is upregulated, affecting fatty acid metabolism when NSAIDs have therapeutic benefits. Giving, that is, a determinant for microinflammation.

Similar to 15-epi-LX biosynthesis, EPA COX-2-derived 15R-HETE was converted by 5-lipoxygenation along with 5 (6) -epoxidation formation in leukocytes to give 15-epi- -LX ₅
It became a series (Fig. 11). C 15-epi-L modified with bulky groups from position 15 to 20
Stable analogs of XA ₄ is not affected by the inactivation enzyme, it inhibits the formation and action of PMN transport and important proinflammatory cytokines (References 10 and 16). Thus, the 5-series 15-epi-LXs have Δ17-182 double bonds, and thus ω-3 derived 15-epi-LXs.
It should act in a similar manner as it can function as an analogue.

The finding was that COX-2-NSAID-dependent oxygenation (eg 18R and 15R) leads to in vivo bioactive compounds that inhibit PMN transendothelial cell migration and invasion.
And ω-3 provide a basis for a novel mechanism of action of nutritional support, namely the generation of an endogenous functional array of lipid signals (Table 1 and Figures 4 and 7), which array plays a key role in microinflammation. Inhibition can mediate some of the beneficial effects observed for omega-3 therapy in human trials. In such a situation, a recognized lipoxygenase product that down-regulates platelet-EC interactions (29. Buchanan, MR, P. Horsewood, and SJ Brister
. 1998. Regulation of endotherial Cell and platelet receptor-ligand bind
ing by the 12-and 15-lipoxygenase monohydroxides, 12-, 15-HETE and 13-HO
DE. Prostaglandins Leukot. Essent. Fatty Acids 58: 339-346) is also produced by COX-2 (Tables 1 and 2) and joins this pathway array and mediator class as in DHA (C22: 6) (Fig. 10C). (Fig. 12) DHA was also converted into a new reaction product (
(Figure 13). In addition, ASA treatment of COX-2 yielded products with different ratios of C17 to C13 (
Figure 14B-G). These new COX-2 products appear to play a role in the cerebrovascular system, where COX-2 and DHA are elevated. Therefore, these compounds can be used for the treatment of inflammatory diseases of nervous tissue (ie Parkinson's disease, Alzheimer's disease, etc.). Thus, surprisingly, the interaction of NSAIDs with COX-2 leads to de novo oxygenation of a wide range of lipid precursors, produces bioactive compounds as illustrated in Figure 15, and can be used in therapeutic treatment. Has been found.

Cardiovascular disease (Ridker, PM Cushman, MJ Stampfer, RP Tracy, and CH
Hennekens. 1997. Inflammation, aspirin, and the risk of cardiovascular d
isease in apparently healthy men. N. Eng. J. Med. 336: 973-979) and PM
These novel compounds ω-3 PUFA processing caused by cell-cell interactions are currently recognized as being associated with inappropriate inflammatory responses in clinical syndromes (ref. 11) affected by N-mediated tissue injury To investigate how pathway identification and the stunning effects of NSAIDs produce potential clinical protection provided by omega-3 PUFA-based recruitment, as well as potent local endogenous mediators controlling microinflammation Develop new means to do so (Fig. 4-11). In addition, the present invention provides potential biochemical indicators and / or markers of basal and effective omega-3 nutritional support to counteract the undesirable effects of current anti-inflammatory therapies.

Table 1. Hydroxy compounds produced from PUFA and recombinant human COX-2 with ASA or COX-2 inhibitors

[0102]

[Table 1]

Results are mean ± SEM, 3. The selective COX-2 inhibitor NS398 was used at 100 uM. All products were extracted, identified and quantified using internal standards and LC / MS / MS. Compounds of interest are shown in bold.

[0104]   Table 2. NSAID-human recombinant COX2 (conversion of n-3 C20: 5)

[0105]

[Table 2]

Values are mean (ng) ± SEM, n = 3. Examples Materials and Methods Zymosan, hematin, NADPH, and ASA were purchased from Sigma-Aldrich. EPA
(Cayman Chemical) and other synthetic standards, hydroxy-fatty acids, and intermediates used for identification were purchased from Cascade Biochem Ltd. B. megaterium is Amer
I purchased it from ican Type Culture Collection. Materials used for liquid chromatography tandem mass spectrometry (LC / MS / MS) are (20. Gronert, K., CB Clish,
M. Romano, and CN Serhan. 1999. Transcellular regulation of eicosanoi
d biosynthesis. In Eicosanoid Protocols. EA Lianos, edited by Humana Press,
Totowa, NJ. 119-144).

Human PMNs were healthy volunteers (drug-free for 2 weeks prior to donation; Brig
Newly isolated from venous blood of Ham and Women's Hospital Protocols no. 88-02642) using a Ficoll gradient and enumerated. Human umbilical vein or microvascular ECs (
HUVECs or HMVECs, respectively) were cultured for transendothelial cell migration (reference 10).
, HMVEC monolayers (1, 2, or 3 subcultures) were seeded (~ 2x10 ⁵ cells / cell) on a polycarbonate permeable support precoated with 0.1% gelatin for incubation with NSAID and PUFA. cm ² ).

Inflammatory exudates were administered to 6-8 week old male FVB mice (bred on standard rodent diet 5001 containing 0.26% n-3 fatty acids) on day 6 of TNF-α (R & D System) (ref. 16), followed by ASA (500 μg) 3.5 hours later, and
Four hours later, 300 μg C20: 5 / capsule was injected. Six hours later, the sac was washed (3 ml saline), the exuded cells were enumerated, and the cells were activated with ₉₄ μM _A23187 at 37 ° C. for 20 minutes. TNF-α stimulation (100 ng / capsule, FVB strain) PMN infiltration by intravenous tail injection of either 18R-hydroxyeicosapentaenoic acid (HEPE), 5,12,18R-HEPE, or 15-epi-LXA analog Inhibition was measured with a bladder lavage fluid collected 4 hours later (reference document 16).

Specific [ ³ H] LTB ₄ , (NEN Life Science Products) binding was performed in human embryonic kidney [HEK] 293 cells stably transfected with human LTB ₄ receptor (Chiang, N. et al.
, K. Groner, CB Clish, JA O'Brien, MW Freeman, and CN Serban. 19
99. Leukotriene B ₄ receptor transgenic mice reveal novel protective role
s for lipoxins and aspirin-triggered lipoxins in reperfusion. J. Clin. I
nvest. 104: 309-316). Human recombinant COX-2 (Dr. RA Copeland, DuPont Merck,
Wilmington, DE). George, HJDE Van Dyk,
RA Straney, JM Trzaskos, and RA Copeland. 1996. Expression purifi
cation and characterization of recombinant human inducible prostaglandin
G / H synthase from baculovirus-infected insect cells. Protein Expres. Pu
7: 19-26. overexpressed in 5/9 insect cells (American Type Culture Collection) by microsomal fraction (~ 8 μl) suspended in Tris (100 mM, pH 8.0) as in rif. Was done. The NSAID PUFA (20 [mu] M) 30 minutes prior to the addition, and incubated (i.e. ASA~1 mM) at 37 ° C., conversion ^1-14 C-labeled C20: 4 (see FIG. 2A and 2B) or C20: 5 (NEN Life Science Products) (see Figures 2C and 2D).

For intermediates and reference compounds, B. megaterium was converted to Bacto Nutrient Broth (F
shaking culture was performed at 30 ° C. in isher Scientific). NADPH (2 mM) and C20: 5 (EPA) in 2 M Tris buffer, pH 8.1 to prepare 18R-HEPE standards.
B. megaterium sonicate incubated with (330 μM) was used for biosynthesis. LTB ₅ (15 μM) was converted to the new product using similar conditions. See results. Incubate was deuterium labeled internal standard (15-HETE and C20: 4)
LC / MS / MS analysis using a Finnigan LCQ equipped with a LUNA C18-2 (150 × 2 mm; 5 μM) column and a rapid spectral scanning UV / Vis detector. In addition, an isocratic eluent (hexane / isopropanol 96: 4 vol /
The R and S alcohol configurations of monohydroxy-PUFA were determined using a Chiralcel CB-H column (JT Baker) using vol). Detailed methods of isolation, quantification, and structure determination of lipid-derived mediators were recently reported and used essentially herein as described for the description of new products.

Production of Hydroxy-DHA Compounds: Hydroxy-DHA compounds were produced in vivo using recombinant COX-II, both in the presence and absence of aspirin acetylation. Briefly, the incubation mixture was prepared using purified recombinant human COX-II as a membrane preparation from SF9 cells expressing the enzyme. Enzyme contains 5 mM phenol
The cells were suspended in 400 μl of 1 M Tris buffer (pH8.0). For aspirin acetylation of COX-II, aspirin (2 mM) was added to the mixture and incubated at 37 ° C for 30 minutes. Then DHA (5 μM) was added and incubated at 37 ° C. for 5 minutes. The reaction was stopped by adding 400 μl of chilled ethanol. The product was then extracted using a solid phase extraction cartridge (SepPak C18).

Novel DHA compounds, 13-hydroxy-DHA, 14-hydroxy-DHA, 16-hydroxy-DHA
, 17-Hydroxy-DHA, 19-Hydroxy-DHA or 20-Hydroxy-DHA had comparable potency to EPA-derived compounds as previously described, eg, the results shown in FIGS. 9A and 9C.

One of ordinary skill in the art would be able to ascertain or ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. These and all other equivalents are intended to be covered by the following claims. All publications and references cited herein, including background portions, are expressly incorporated herein by reference in their entirety.

[Brief description of drawings]

The present invention will be more fully understood from the following detailed description in conjunction with the accompanying drawings.

FIG. 1 depicts transcellular lipid mediator (LM) biosynthesis.

FIG. 2A shows a thin layer chromatogram of the product produced from ¹⁴ C-labeled arachidonic acid (AA, ω-6) by cyclooxygenase 2 (COX-2). Figure 2B shows ¹⁴ C-labeled arachidonic acid (AA, ω-6) with aspirin acetylated COX-2.
2 represents a thin layer chromatogram of the product generated from FIG. 2C represents a thin layer chromatogram of the product produced from ¹⁴ C-labeled eicosapentaenoic acid (EPA, ω-3) by cyclooxygenase II (COX-2). FIG. 2B represents a thin layer chromatogram of the product produced from ¹⁴ C-labeled EPA (ω-3) by aspirin acetylated-COX-2.

FIG. 3 shows the conversion of EPA (ω-3) by B. megaterium by LC / MS ion chromatogram and mass spectral analysis.

FIG. 4A shows EPA in dorsal air pouch inflammatory exudate of aspirin-treated mice.
3 is an LC / MS ion chromatogram of the monohydroxy product produced from FIG. 4B is a mass spectral analysis of 18R-HEPE produced from EPA in the dorsal air pouch inflammation exudate of aspirin-treated mice. FIG. 4C is a mass spectral analysis of 5S-HEPE produced from EPA in the dorsal air pouch inflammation exudate of aspirin-treated mice. FIG. 4D is a mass spectral analysis of 5,12,18R-tri-HEPE produced from EPA in the dorsal air pouch inflammation exudate of aspirin-treated mice.

FIG. 5 is LC / LC of the 5,12,18-triHEPE isomer produced from 5S, 12R-dihydroxy-6Z, 8E, 14Z, 17Z-eicosapentaenoic acid (ω-3) by B. megaterium. MS ion chromatogram is represented with mass spectral analysis.

FIG. 6 represents mouse dorsal air pouch treated with tumor necrosis factor-alpha (TNF-α) and aspirin.

FIG. 7A represents an LC / MS ion chromatogram of 18-HEPE generated from IL-1β-stimulated human umbilical cord endothelial cells (HUVEC) treated with aspirin and EPA. Figure 7B shows aspirin acetylation-COX-2 and serum treated zymosan (
STZ) -represents an LC / MS ion chromatogram of avian HEPEs produced from EPA by stimulated human PMN. FIG. 7C represents a mass spectral analysis of the tri-HEPE product I, 5,12,18R-tri-HEPE in FIG. 7B. FIG. 7D represents a mass spectral analysis of the tri-HEPE product II, 15-epi-LXA ₅ in FIG. 7B.

FIG. 8 is a selective ion monitoring LC / MS / MS chromatogram of the monohydroxy product produced from EPA by aspirin acetylation-COX-2 and 18
-Represents mass spectral analysis of -HEPE, 15-HEPE and 11-HEPE.

FIG. 9A shows LTB ₄ -stimulated PMN transendothelial cell migration by 18-HEPE (○), 15,12,18R-tri-HEPE (●) and aspirin-induced lipoxin (ATL) analog reference compound (■). Shows inhibition. FIG. 9B shows 18R-HEPE using ³ H-LTB ₄ (○), 5,12,18R-tri-HEPE (●), LTB against recombinant human LTB ₄ receptor stably expressed in HEK-293 cells. ₅ shows competitive binding between ₅ (□) or homoligand LTB ₄ (■). FIG. 9C shows 18R-HEPE, 5,12,18-tri-HEPE, or the ATL analog reference compound used for comparison (analog 15 (S) -16 (para-fluoro) -phenoxy-LTB ₄ Inhibition of TNF-α-induced leukocyte trafficking into the mouse dorsal air pouch after intravenous injection of either 100 ng, results represent N = 4.

FIG. 10A is an LC / MS / MS chromatogram and mass spectral analysis of the monohydroxy product produced from dihomo-γ-linoleic acid (C20: 3, ω-3) and aspirin acetylated-COX-2. Represents FIG. 10B represents an LC / MS / MS chromatogram and mass spectral analysis of the monohydroxy product produced from linolenic acid (C18: 3, ω-3) and aspirin acetylated-COX-2. FIG. 10C represents an LC / MS / MS chromatogram and mass spectral analysis of the monohydroxy product produced from linoleic acid (C18: 2, ω-6) and aspirin acetylated-COX-2.

FIG. 11. Proposed Scheme for Generating Functional Arrays of Lipid Signals from ω-3 PUFAs by Transcellular Processing: Endogenous Inhibitors of Microinflammation. At sites where COX-2 is upregulated and treated with NSAIDs, prostaglandin formation from C20: 4 is inhibited. Systemic ω-3 PUFAs are converted by the COX-2-NSAID lipoxygenase-type mechanism to stereospecifically extract hydrogen with EPA (C20: 5) (panel A) C16 or C13, and to release the molecule O ₂ . R insert and 15R-H (p) EPE or 18 from epoxide
Produces RH (p) EPE or is reduced to alcohol, or similarly DHA
(At (C22: 6) C13 or C17 (panel B), the molecule O ₂ is inserted by R insertion and 13-hydroxy-DH
Generates A or 17-hydroxy-DHA. The full stereochemistry of the trihydroxy compound remains to be determined and is indicated in the appropriate configuration. These compounds interact with cells in the local microenvironment and inhibit PMN recruitment. COX-2-NSAID-dependent hydrogen extraction and molecular oxygen insertion are all ω-3 PUFs with 1,4-cispentadiene units
Found in A.

FIG. 12 depicts an aspirin acetylation-COX-2-dependent pathway for the production of novel lipid mediators that are also markers of aspirin treatment.

FIG. 13 depicts the structures of the major and minor products produced from docosahexaenoic acid (DHA, C22: 6, ω-3) and aspirin acetylated-COX-2.

FIG. 14A shows DH induced by acetylation of COX-2 by aspirin.
A represents the conversion of the spot color of the product. The LC / MS ion chromatogram in the left panel shows that 13-hydroxy-DHA is the major product in the absence of aspirin. Acetylation of COX-2 by aspirin suppresses 13-hydroxy-DHA production and 17-hydroxy-DHA production becomes the major product as shown in the right panel. Figure 14B shows aspirin acetylation- 13-hydroxy-generated from DHA by COX-2.
Figure 2 represents LC / MS / MS ion chromatogram and mass spectral analysis of DHA. Figure 14C shows 14-hydroxy-produced from DHA by aspirin acetylation-COX-2.
Figure 2 represents LC / MS / MS ion chromatogram and mass spectral analysis of DHA. Figure 14D shows 16-hydroxy-produced from DHA by aspirin acetylation-COX-2.
Figure 2 represents LC / MS / MS ion chromatogram and mass spectral analysis of DHA. FIG. 14E shows 17-hydroxy-produced from DHA by aspirin acetylation-COX-2.
Figure 2 represents LC / MS / MS ion chromatogram and mass spectral analysis of DHA. FIG. 14F shows 19-hydroxy-produced from DHA by aspirin acetylation-COX-2.
Figure 2 represents LC / MS / MS ion chromatogram and mass spectral analysis of DHA. FIG. 14G shows 20-hydroxy-produced from DHA by aspirin acetylation-COX-2.
Figure 2 represents LC / MS / MS ion chromatogram and mass spectral analysis of DHA.

FIG. 15 shows the site selectivity for oxygenation of ω-3 and ω-6 by aspirin acetylation-COX-2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 11/00 A61P 11/00 11/06 11/06 17/04 17/04 17/06 17/06 17 / 08 17/08 19/02 19/02 27/02 27/02 29/00 29/00 37/08 37/08 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA , ZWF F-term (reference) 4C086 AA01 AA02 AA03 DA17 MA02 NA05 NA14 ZA33 ZA36 ZA40 ZA59 ZA89 ZA96 ZB11 ZB13 4C206 DA07 MA02 NA05 NA14 ZA33 ZA36 ZA38 ZA40 ZA59 ZA89 ZA96 ZB11 A23A23 A006

Claims (39)

[Claims] 1. The following formula: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 2. The compound of claim 1, wherein the C-15 carbon has the R configuration. 3. The compound of claim 1, wherein the C-15 carbon has the S configuration. 4. The following formula: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 5. The compound of claim 4, wherein the C-18 carbon has the R configuration. 6. The compound of claim 4, wherein the C-18 carbon has the S configuration. 7. The following formula: embedded image Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and one or more P is a hydrogen atom or one or more protecting groups, or a combination thereof. 8. A C-5 carbon has an S configuration, a C-12 carbon has an R configuration, and a C-1
The compound of claim 7, wherein 8 carbons have the R configuration. 9. The following formula: embedded image Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and one or more P is a hydrogen atom or one or more protecting groups, or a combination thereof. 10. A C-5 carbon has an S configuration, a C-6 carbon has an R configuration, and a C-
The compound of claim 7, wherein 15 carbons have the R configuration. 11. Omega-3 so that inflammation is treated or prevented in a subject.
A method for treating or preventing inflammation in such a subject comprising administering a combination of a fatty acid and aspirin. 12. The method of claim 11, wherein the omega-3 fatty acid and aspirin are administered at two different times. 13. The method of claim 11, wherein the omega-3 fatty acid is eicosapentaenoic acid. 14. The method of claim 11, wherein the omega-3 fatty acid is docosahexaenoic acid. 15. An arterial inflammation in a subject, comprising the step of administering to the subject a combination of omega-3 fatty acids and aspirin, such that the arterial inflammation, arthritis, or cardiovascular disease is treated or prevented in the subject. A method for treating or preventing arthritis, or cardiovascular disease. 16. The method of claim 15, wherein the omega-3 fatty acid and aspirin are administered at two different times. 17. The method of claim 15, wherein the omega-3 fatty acid is eicosapentaenoic acid. 18. The method of claim 15, wherein the omega-3 fatty acid is docosahexaenoic acid. 19. The following formula so that inflammation is treated or prevented in a subject: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug and P is a hydrogen atom or a protecting group A method for treating or preventing inflammation. 20. The following formula so that inflammation is treated or prevented in a subject: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation. 21. The following formula so that inflammation is treated or prevented in a subject: Where R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and wherein one or more P is a hydrogen atom or one or more protecting groups or combinations thereof. A method for treating or preventing inflammation in such a subject, comprising the step of: administering to the subject. 22. The following formula so that inflammation is treated or prevented in a subject: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and one or more P is a hydrogen atom or one or more protecting groups or a combination thereof. A method for treating or preventing inflammation in such a subject, comprising the step of: administering to the subject. 23. The following formula so that arterial inflammation, arthritis, or cardiovascular disease is treated or prevented in a subject: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating arterial inflammation, arthritis, or cardiovascular disease. 24. The following formula so that arterial inflammation, arthritis, or cardiovascular disease is treated or prevented in a subject: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, including administering to the subject an arterial inflammation,
A method for treating arthritis, or cardiovascular disease. 25. In order to treat or prevent arterial inflammation, arthritis, or cardiovascular disease in a subject, the following formula: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and one or more P is a hydrogen atom or one or more protecting groups or a combination thereof to a mammal. A method for treating arterial inflammation, arthritis, or cardiovascular disease in such a subject, comprising administering. 26. The following formula so that arterial inflammation, arthritis, or cardiovascular disease is treated or prevented in a subject: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and one or more P is a hydrogen atom or one or more protecting groups or a combination thereof to a mammal. A method for treating arterial inflammation, arthritis, or cardiovascular disease in such a subject, comprising administering. 27. Hydroxyl-protected or unprotected monohydroxyl-docosahexaenoic acid or a pharmaceutically acceptable analogue thereof. 28. The following formula: embedded image Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 29. The following formula: embedded image Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 30. The following formula: embedded image Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 31. The following formula: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 32. The following formula: embedded image Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 33. The following formula: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group. 34. The following formula so that the subject is treated: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation. 35. The following formula so that the subject is treated: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation. 36. The following formula so that the subject is treated: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation. 37. The following formula so that the subject is treated: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation. 38. The following formula so that the subject is treated: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation. 39. The following formula so that the subject is treated: Wherein R is a hydrogen atom or a pharmaceutically acceptable salt, ester, amide or prodrug, and P is a hydrogen atom or a protecting group, in the subject A method for treating or preventing inflammation.